P465: A LIPID NANOPARTICLE DELIVERY SYSTEM FOR TARGETING THE LEUKAEMIC FUSION GENE RUNX1/ETO BY SIRNA
Authors: L. Swart, P. Derevyanko, A. van Oort, M. Ashtiani, A. Krippner-Heidenreich, A. Koekman, C. Seinen, H. Issa, H. Blair, R. Schiffelers, O. Heidenreich
Journal: HemaSphere (2022)

Background: The leukaemic fusion gene RUNX1/ETO initiates and drives leukaemogenesis and, thus, constitutes an ideal cancer-specific target. Direct and exclusive targeting of leukaemic fusion proteins using RNA interference is an attractive concept, but has proven challenging because of the unfavourable pharmacokinetic properties of siRNA. To address this challenge, we have developed lipid nanoparticle (LNP) formulations that interfere with the expression in leukaemic cell lines and in primary cells. To further harness their use in patient-derived AML cells, we are exploring and optimizing targeted LNPs for effective siRNA delivery to primary leukaemic cells. Aims: To develop and validate a delivery system for the therapeutic application of fusion gene-specific siRNAs. Methods: LNPs encapsulating siRE (active siRNA targeting the RUNX1/ETO fusion transcript) and siMM (mismatch siRNA control with two nucleotides swapped in the sense strand) were prepared using microfluidic mixing. Ligand or fluorescent dyes were conjugated to polyethylene glycol (PEG) following a copper-free click chemistry approach. LNPs were characterized by dynamic light scattering (DLS) and cryo electron microscopy (EM). Functional experiments with patient-derived t(8;21) cells were performed in co-culture with mesenchymal stromal cells under conditions that preserved and expanded immature VLA-4 expressing leukaemic populations as determined by an 11 antibody panel and flow cytometry analysis. Transcript and protein levels of RUNX1/ETO and target genes were determined by bulk and single cell (sc) RNA-seq, real-time polymerase chain reaction and western blotting. Results: To monitor uptake of 50 – 60 nanometers sized LNPsiRNA we labelled the surface with Cy3 and a ligand by a PEG post-insertion approach. To improve uptake and tissue retention, we chose a modified short peptide ligand harbouring an LDV motif with high affinity towards the very late antigen-4 (VLA-4) that is widely expressed across haematopoietic cells. Decoration of the LNPs with the LDV resulted in an enhanced uptake in t(8;21) cell lines within the first 8 hours. While undecorated LNPs were hardly taken up by patient-derived material, the decoration with LDV resulted in an efficient uptake and a concomitant knockdown of RUNX1/ETO transcript and protein after a single dose. Sequential treatment leads to longer-lasting reduction of RUNX1/ETO transcript and associated target genes (LAPTM5, CCND2, C/EBPA and ANGPT1) and loss of RUNX1/ETO protein. Combined multiparameter flow analysis and scRNAseq of patient cells show that RUNX1/ETO depletion causes massive shifts in cell populations and an almost complete loss of immature populations (CD34+ CD38- CD117+ CD45RA+) associated with a gain in mature (CD15+) populations, indicating a release from the differentiation block caused by the fusion protein. Gene Set Enrichment Analysis (GSEA) of scRNAseq data confirm the role of RUNX1/ETO in promoting stemness and cell cycle progression and highlight a key function of this fusion protein in coordinating metabolism in support of leukaemic propagation. Currently, we are performing in vivo biodistribution and efficacy studies using orthotopic mouse models. Summary/Conclusion: Decoration of LNPs with an LDV motif enhances uptake of fusion transcript-specific siRNAs and enables efficacious knockdown of RUNX1/ETO in patient-derived cells. The biologic consequences validate RUNX1/ETO as a highly promising and leukaemia-specific target and highlight the LNP-mediated delivery of siRNAs as a promising new approach for the treatment of fusion gene-driven leukaemias.